从匹兹堡基金会和国家卫生研究院（NIH）的[GM087798; CA148629; ES019498] RWS的赠款，以支持这项工作。 UPCI慢病毒设施的圣淘沙名胜世界提供了支持，从国家卫生研究院癌症研究中心支援津贴[P30的CA047904]。由美国匹兹堡大学的药理学部化学生物学的DS也提供了支持。也提供了支持ESTRO到CV。

<ol>
	<li>Mutamba, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XRCC1+and+base+excision+repair+balance+in+response+to+nitric+oxide.">XRCC1 and base excision repair balance in response to nitric oxide.</a> DNA Repair (Amst). 10, 1282-1293 (2011).</li><li>Tang, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-methylpurine+DNA+glycosylase+and+DNA+polymerase+beta+modulate+BER+inhibitor+potentiation+of+glioma+cells+to+temozolomide.">N-methylpurine DNA glycosylase and DNA polymerase beta modulate BER inhibitor potentiation of glioma cells to temozolomide.</a> Neuro-oncology. 13, 471-486 (2011).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+and+nucleic+acids.">Fluorescence resonance energy transfer and nucleic acids.</a> Methods Enzymol. 211, 353-388 (1992).</li><li>Yaron, A., Carmel, A., Katchalski-Katzir, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramolecularly+quenched+fluorogenic+substrates+for+hydrolytic+enzymes.">Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes.</a> Anal. Biochem. 95, 228-235 (1979).</li><li>Kreklau, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+fluorometric+oligonucleotide+assay+to+measure+O(+6)-methylguanine+DNA+methyltransferase,+methylpurine+DNA+glycosylase,+8-oxoguanine+DNA+glycosylase+and+abasic+endonuclease+activities:+DNA+repair+status+in+human+breast+carcinoma+cells+overexpressing+methylpurine+DNA+glycosylase.">A novel fluorometric oligonucleotide assay to measure O( 6)-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase.</a> Nucleic Acids Res. 29, 2558-2566 (2001).</li><li>Belon, C. A., Frick, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+helicase+activity+with+molecular+beacons.">Monitoring helicase activity with molecular beacons.</a> BioTechniques. 45, 433-442 (2008).</li><li>Ma, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+DNA+polymerase+activity+using+molecular+beacon.">Real-time monitoring of DNA polymerase activity using molecular beacon.</a> Anal. Biochem. 353, 141-143 (2006).</li><li>Summerer, D., Marx, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+beacon+for+quantitative+monitoring+of+the+DNA+polymerase+reaction+in+real-time.">A molecular beacon for quantitative monitoring of the DNA polymerase reaction in real-time.</a> Angew. Chem. Int. Ed. Engl. 41, 3620-3622 (2002).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+molecular+beacon+to+monitor+activity+of+E.+coli+DNA+ligase.">Using molecular beacon to monitor activity of E. coli DNA ligase.</a> The Analyst. 130, 350-357 (2005).</li><li>Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+beacons+for+the+detection+of+DNA+and+telomerase+activity.">Catalytic beacons for the detection of DNA and telomerase activity.</a> J. Am. Chem. Soc. 126, 7430-7431 (2004).</li><li>Kundu, L. M., Burgdorf, L. T., Kleiner, O., Batschauer, A., Carell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavable+substrate+containing+molecular+beacons+for+the+quantification+of+DNA-photolyase+activity.">Cleavable substrate containing molecular beacons for the quantification of DNA-photolyase activity.</a> Chembiochem : a European journal of chemical biology. 3, 1053-1060 (2002).</li><li>Sokol, D. L., Zhang, X., Lu, P., Gewirtz, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+detection+of+DNA.RNA+hybridization+in+living+cells.">Real time detection of DNA.RNA hybridization in living cells.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 11538-11543 (1998).</li><li>Yang, X., Tong, C., Long, Y., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+fluorescence+assay+for+hSMUG1+activity+based+on+modified+molecular+beacon.">A rapid fluorescence assay for hSMUG1 activity based on modified molecular beacon.</a> Molecular and cellular probes. 25, 219-221 (2011).</li><li>Mirbahai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+molecular+beacon+to+track+the+activity+of+base+excision+repair+protein+OGG1+in+live+cells.">Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.</a> DNA Repair (Amst). , (2009).</li><li>Maksimenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+beacon+assay+for+measuring+base+excision+repair+activities.">A molecular beacon assay for measuring base excision repair activities.</a> Biochem. Biophys. Res. Commun. 319, 240-246 (2004).</li></ol>

RWS的是一个科学顾问公司Trevigen，其余作者的声明，有没有利益冲突。

有超过600个使用“分子信标”检测和量化许多分子和酶的活动，包括解旋酶的活性，DNA聚合酶活性7,8，DNA连接酶的活动9，10端粒酶活性，DNA光解酶的活性11和DNA /引用许多人的RNA之间的杂交种12。除了 ​​利用分子信标测量DNA修复上面列出的活动，我们和其他人已经证明误码活动1,2,13-15分析这些探头的效用。 实时BER活性测定，我们描述...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>氯化钾</td><td>费舍尔</td><td> P217-500 </td><td>分子级</td></tr><tr><td> EDTA的</td><td>费舍尔</td><td> BP120-500 </td><td></td></tr><tr><td>甘油</td><td>费舍尔</td><td> BP229-1 </td><td>分子级</td></tr><tr><td>数码地面电视</td><td>费舍尔</td><td> BP172-5 </td><td></td></tr><tr><td>肝素钠解决方案</td><td> Invitrogen公司</td><td> 15630 </td><td></td></tr><tr><td>肝素钠的盐</td><td>西格玛</td><td> H4034-500G </td><td></td></tr><tr><td>氢氧化钾</td><td>西格玛</td><td> 17-8 </td><td></td></tr><tr><td> 0.22μm的过滤器</tD&gt; <td> NALGENE </td><td> 167-0020 </td><td></td></tr><tr><td>幻灯片一仪透析产品</td><td>刺穿</td><td> 66373 </td><td>兆瓦7000 </td></tr><tr><td>注射器</td><td>费舍尔</td><td> 309659 </td><td></td></tr><tr><td>针</td><td>费舍尔</td><td> 305196 </td><td></td></tr><tr><td> 1.5毫升离心管</td><td>费舍尔</td><td> 05-408-129 </td><td>黑色</td></tr><tr><td>猎鹰管15毫升</td><td>费舍尔</td><td> 352097 </td><td></td></tr><tr><td> NucBus​​ter蛋白提取试剂盒</td><td> Calbiochem公司</td><td> 71183 </td><td></td></tr><tr><td> PBS </td><td> Invitrogen公司</td><td> 14190 </td><td></td></tr><tr><td>分子信标</td><td>内幕交易审裁处</td><td></td><td></td></tr><tr><td> BioRad公司蛋白检测染料重新代理精矿</td><td> BioRad公司</td><td> CAT＃500-0006 </td><td></td></tr></tbody></table>

quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons

我们描述了一个定量，实时测量碱基切除修复（BER）的分子信标DNA糖基化酶和AP内切酶的活动，在核细胞裂解方法。基板（灯塔）是由deoxyoligonucleotide包含单碱基损伤与6 - 羧基荧光素（6-FAM）基团结合到5&#39;端和DABCYL基的共轭寡核苷酸的3&#39;结束。 BER分子信标是43基地在长度和序列的目的是为了促进与13环和15个碱基对中干1,2个核苷酸的茎环结构的形成。在此配置折叠时6-FAM基淬火DABCYL非荧光的方式通过福斯特共振能量转移（FRET）3,4。病变位置等基地，以下病灶切除和链的断裂，其余5个基地的寡核苷酸，含6-FAM基干公布。释放第二支队从猝灭（DABCYL）荧光的增加，DNA修复水平是相称的结果。通过收集多个读取的荧光值，实时误码活动的评估是可能的。标准定量实时PCR仪器的使用，使许多样品同时分析。这些BER分子信标设计，用一个单一的基础病变，是适合进行动力学分析，误码率的量化和抑制剂验证，是适应组织和肿瘤细胞裂解物中的DNA修复能力的量化或用纯化的蛋白质。在肿瘤裂解物或使用这些分子信标组织抽吸的BER活动的分析，可能是适用于功能性的生物标志物的测量。此外，使用这种定量检测与纯化蛋白质的BER活动分析，误码率抑制剂的发现和验证提供了一种快速，高通量的方法。

Watch this Scientific Journal Video about Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons at JoVE.com

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

我们描述了DNA糖基化酶和AP内切酶的活动，在核细胞裂解定量，实时测量的方法。该法产生服从动力学分析DNA修复活动率，是适应组织和肿瘤裂解物或纯化蛋白质的DNA修复能力的量化。

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

quantitative-real-time-analysis-base-excision-repair-activity-cell

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Biology

13.5K 次观看.  University of Pittsburgh School of Medicine. 我们描述了DNA糖基化酶和AP内切酶的活动，在核细胞裂解定量，实时测量的方法。该法产生服从动力学分析DNA修复活动率，是适应组织和肿瘤裂解物或纯化蛋白质的DNA修复能力的量化。

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

定量，实时分析，利用分子信标病变特定的细胞裂解液中的碱基切除修复活动